Method and primers for detecting viral genes

ABSTRACT

A method for detecting luteoviruses comprises extracting nucleic acid from plant tissue and making a first strand DNA using a PCR primer and amplifying the DNA using a second PCR primer to obtain as PCR product and comparing amino acid sequences in the PCR product obtained with known amino acid sequences characteristic of the luteovirus to detect the presence of the luteovirus.

TECHNICAL FIELD

[0001] The present invention relates to a method for the detection of luteoviruses using polymerase chain reaction (PCR) primers and to amino acid sequences which can be used to design the PCR primers. In particular, the invention relates to methods and primers for detection of carrot red leaf luteovirus (CRLV).

PRIOR ART

[0002] Carrot motley dwarf is a serious viral disease of carrots and other umbelliferous plants such as parsley and chervil. Symptoms include red and yellow discolouration of leaves and stunting. In general, luteoviruses can be spread efficiently by aphids, cause serious disease in vegetables and cereals and are of global economic importance.

[0003] Carrot motley dwarf (CMD) is an aphid transmissible virus disease caused by the combination of the two types of virus (carrot red leaf luteovirus (CRLV) and carrot mottle umbravirus (CMoV)). Although each of these viruses are independently pathogenic (Watson, M., Serjeant, E. P., and Lennon, E. A. (1964) Annals of Applied Biology 54;153-166), the umbravirus component can only be transmitted by aphids when encapsidated by luteovirus-coded coat protein (Elnagar, S., and Murant, A. F. (1978) Annals of Applied Biology 89, 237-244.; Waterhouse, P. M., and Murant, A. F. (1983) Annals of Applied Biology 103;455-464). Luteoviruses are largely phloem restricted (Murant, A. F., Roberts, I. M. (1979). Virus-like particles in phloem tissue of chervil (Anthriscus cerefolium) infected with carrot red leaf luteovirus. Annals of Applied Biology 92;343-346) whereas umbraviruses are able to spread systemically throughout the plant. Luteoviruses are able to gain from association with CMoV because CMoV facilitates luteovirus accumulation (Barker, H. (1989) Ann.appl.Biol 115;71-78), and though each can persist alone, the combination has a potential for greater fitness. Other examples of luteoviruses include potato leaf roll virus (PLRV), beet western yellows virus (BWYV) and barley yellow dwarf virus (BYDV).

[0004] There is to date no rapid and sensitive detection technique for CMD viruses. It is thought that this could be due to several reasons, firstly, the carrot mottle component of CMD cannot easily be detected serologically as the virus has no coat protein. In addition, a high level of genetic variation is proposed to exist among carrot mottle virus isolates, making molecular detection difficult. The carrot red leaf component of CMD can be detected serologically, but the virus accumulates only in the phloem of infected plants and only at low levels.

[0005] Robertson, N. L. French, R.Gray S. M (1991) J. Gen. Virol. 72:1473-1477 disclose a PCR based method using a single luteovirus primer pair to diagnose a range of luteoviruses including five barley yellow dwarf luteovirus serotypes by one assay using the luteoviral coat protein gene sequences to design PCR primers. However the primer pairs disclosed in this document are unable to detect CRLV.

[0006] Vercruysse et al (2000, J. Virological Methods 88:153-161) describes the design of a pair of redundant primers designed to target conserved regions of the RdRp genes from an unpublished partial sequence of a Canadian isolate of CRLV and sequences from five potato leafroll polerovirus (PLRV) isolates. The primers were able to identify cDNA from CRLV and PLRV. However, the primers disclosed by this document were not able to detect luteoviruses other than CRLV and PLRV.

[0007] Therefore, although there exists a diagnostic assay which is able to identify CRLV infection, there is a need for a single broad spectrum diagnostic assay which is capable of diagnosing a large number of luteoviruses including the agronomically important CRLV. Thus it can be seen that a luteovirus detection system capable of detecting a broad spectrum of luteoviruses including CRLV would provide a contribution to the art.

DISCLOSURE OF THE INVENTION

[0008] The present inventors have now devised primers capable of detecting the presence of a number of luteoviruses including CRLV sequence. In particular, a previously unpublished CRLV nucleic acid sequence has been aligned with nucleic acid sequences of a number of other luteoviruses and primers have been designed to conserved regions of the sequence corresponding to regions of high degeneracy in the translated amino acid sequence.

[0009] Accordingly, there is provided in a first aspect of the present invention, a primer capable of hybridising to conserved regions of nucleic acid of carrot red leaf luteovirus (CRLV), potato leafroll polerovirus (PLRV) and at least one of barley yellow dwarf virus (BYDV) and beet western yellow virus (BWYV). In preferred embodiments, the primer is capable of hybridising to conserved regions of nucleic acid of carrot red leaf luteovirus (CRLV), potato leafroll polerovirus (PLRV), barley yellow dwarf virus (BYDV) and beet western yellow virus (BWYV).

[0010] Oligonucleotide primers may be designed on the basis of amino acid sequence information, taking into account the degeneracy of the genetic code, and, where appropriate, codon usage of the organism from which the candidate nucleic acid is derived. An oligonucleotide for use in nucleic acid amplification may have about 10 or fewer codons (e.g. 6, 7 or 8), i.e. be about 30 or fewer nucleotides in length (e.g. 18, 21 or 24). Generally specific primers are upwards of 14 nucleotides in length, but need not be longer than 18-20. Those skilled in the art are well versed in the design of primers for use in processes such as PCR. Various techniques for synthesizing oligonucleotide primers are well known in the art, including phosphotriester and phosphodiester synthesis methods.

[0011] Preferred amino acid sequences suitable for use in the design of probes or PCR primers may include conserved (completely, substantially or partly) regions of sequences corresponding to a RNA dependent RNA polymerase (RdRp). In one embodiment, the PCR primers which can be used can be designed by obtaining the nucleotide sequence corresponding to a RNA dependent RNA polymerase (RdRp) gene in an isolate of CRLV, making alignments of putative RNA-dependent RNA polymerase (RdRp) proteins of BWYV, PLRV and BYDV, and the predicted amino acid sequence of CRLV and using the regions of amino acid conservation to design PCR primers.

[0012] The CRLV sequence is preferably all or part of the CRLV sequence of FIG. 2.

[0013] Such sequences include the FVKGEPH, GFKVEV, EDDMEV or KGEPHK motifs of the CLRV amino acid sequence of FIG. 2.

[0014] Thus preferred primers according to the invention include primers which encode the amino acid sequence FVKGEPH (SEQ ID NO:1) (5′ TTY/GTN/AAR/GGN/GAR/CCN/CAY 3′ (CL1, SEQ ID NO:2)); primers which hybridise to the nucleic acid encoding amino acid sequence GFKVEV (SEQ ID NO:3) (5′NAC/YTC/NAC/YTT/RAA/NCC 3′ (CL2, SEQ ID NO:4)) and primers which hybridise to the nucleic acid encoding amino acid sequence EDDMEV (SEQ ID NO:5) (5′ CK/NAC/YTC/CAT/RTC/RTC/YTC 3′ (CL3, SEQ ID NO:6)). As an alternative to the CL1 primer disclosed above, a primer which encodes the amino acid sequence KGEPHK (SEQ ID NO:7) (5′ AAR/GGN/GAR/CCN/CA Y/AAR/C 3′ (CL4, SEQ ID NO:8)) may also be used.

[0015] In the context of the present invention, “Y”, “R”, “N” and “K” take their normal meanings i.e. “Y”=T/U or C, “R”=G or A, “N”=A or G or C or T/U and “K”=G or T/U.

[0016] Uses of Primers

[0017] Primers of the invention may be used to diagnose the presence of a luteovirus sequence in a target nucleic acid sample.

[0018] Thus in a further aspect of the invention, there is provided a method for diagnosing the presence of a luteovirus sequence in a plant tissue nucleic acid sample, the method comprising the step of: hybridising a primer according to the invention to the nucleic acid.

[0019] A luteovirus nucleic acid sequence identified using a method of the invention may be probed under conditions for selective hybridisation and/or subjected to a specific nucleic acid amplification reaction such as the polymerase chain reaction (PCR) (reviewed for instance in “PCR protocols; A Guide to Methods and Applications”, Eds. Innis et al, 1990, Academic Press, New York, Mullis et al, Cold Spring Harbor Symp. Quant. Biol., 51:263, (1987), Ehrlich (ed), PCR technology, Stockton Press, NY, 1989, and Ehrlich et al, Science, 252:1643-1650, (1991)). PCR comprises steps of denaturation of template nucleic acid (if double-stranded), annealing of primer to target, and polymerisation. The nucleic acid probed or used as template in the amplification reaction may be genomic DNA, cDNA or RNA. Other specific nucleic acid amplification techniques include strand displacement activation, the QB replicase system, the repair chain reaction, the ligase chain reaction and ligation activated transcription. For convenience, and because it is generally preferred, the term PCR is used herein in contexts where other nucleic acid amplification techniques may be applied by those skilled in the art. Unless the context requires otherwise, reference to PCR should be taken to cover use of any suitable nucleic amplification reaction available in the art.

[0020] A method may include hybridisation of one or more (e.g. two) probes or primers to target nucleic acid. Where the nucleic acid is double-stranded DNA, hybridisation will generally be preceded by denaturation to produce single-stranded DNA. The hybridisation may be as part of a PCR procedure, or as part of a probing procedure not involving PCR. An example procedure would be a combination of PCR and low stringency hybridisation. A screening procedure, chosen from the many available to those skilled in the art, is used to identify successful hybridisation events and isolated hybridised nucleic acid.

[0021] Binding of a probe or primer to target nucleic acid (e.g. DNA) may be measured using any of a variety of techniques at the disposal of those skilled in the art. For instance, probes may be radioactively, fluorescently or enzymatically labelled. Other methods not employing labelling of probe include examination of restriction fragment length polymorphisms, amplification using PCR, RN'ase cleavage and allele specific oligonucleotide probing. Probing may employ the standard Southern blotting technique. For instance DNA may be extracted from cells and digested with different restriction enzymes. Restriction fragments may then be separated by electrophoresis on an agarose gel, before denaturation and transfer to a nitrocellulose filter. Labelled probe may be hybridised to the DNA fragments on the filter and binding determined. DNA for probing may be prepared from RNA preparations from cells.

[0022] In a preferred embodiment of the invention, the method comprises the steps of treating nucleic acid with a first PCR primer of the invention to obtain a DNA product; treating the DNA product with one or more second PCR primers according to the invention to obtain a PCR product; and comparing the nucleotide sequence or corresponding amino acid sequence of the PCR product with a known nucleotide sequence or corresponding amino acid sequence characteristic of the luteovirus.

[0023] In one embodiment of the invention, RT-PCR is carried out using CL2 as a cDNA primer and a mixture of CL1 and CL2 as PCR primers. This set of primers is capable of detecting a broad range of luteoviruses including PLRV, CRLV, beet western yellows virus (BWYV) and beet mild yellowing virus (BMYV).

[0024] Where a sample is identified as comprising a luteovirus sequence using the method of the invention, PLRV and CRLV may be discriminated from other luteoviruses such as BWYV and BMYV by repeating the method of the invention, but using CL1 and CL3 as the PCR primers. If luteovirus is detected using CL2 as the cDNA primer and a mixture of CL1 and CL3 as PCR primers, the luteovirus must be CRLV or PLRV.

[0025] Thus, in a further aspect of the present invention, there is provided a method for diagnosing the presence of at least one of CRLV or PLRV sequences in a plant tissue, the method comprising the steps of:

[0026] providing nucleic acid from plant tissue;

[0027] treating the nucleic acid with a PCR primer CL2 to obtain a DNA product;

[0028] treating the DNA product with PCR primers CL1 and CL3 to obtain a PCR product; and

[0029] comparing a nucleotide sequence or corresponding amino acid sequence in the PCR product with a nucleotide sequence or corresponding amino acid sequence characteristic of the luteovirus to detect the presence of the luteovirus.

[0030] The luteovirus sequences may be present due to pathological infection of the plant or may be present as a transgene introduced by recombinant DNA technology. Luteovirus sequences have been introduced as transgenes for crop protection (for example see WO 9418336, Monsanto).

[0031] Kits

[0032] A primer or primers according to the invention may be provided as part of a kit, e.g. in a suitable container such as a vial in which the contents are protected from the external environment. The kit may include instructions for use of the nucleic acid, e.g. in PCR and/or a method for determining the presence of nucleic acid of interest in a test sample. A kit wherein the nucleic acid is intended for use in PCR may include one or more other reagents required for the reaction, such as polymerase, nucleosides, buffer solution etc. The nucleic acid may be labelled. A kit for use in determining the presence or absence of nucleic acid of interest may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself. Such kits form another aspect of the present invention.

[0033] In preferred embodiments of the invention the kit includes one or more primers for the determination of the presence of the umbravirus CMoV. Preferred CMoV primers include the carmovirus related primers Carmo-2 and Carmo-6, the CMoV specific primer NC1 or the CMoV specific primer NC-2. Indeed such primers form an independent aspect of the present invention.

[0034] The carmovirus related primer Carmo-6 is based on the amino acid sequence VCDDGNN (SEQ ID NO:11); the deduced nucleic acid sequence is:

G(AC)(AC)CTGCAG(AGCT)AC(AG)CA(AG)TC(AG)TC(AGCT)CC(AG)TT(AG)TT   (SEQ ID NO:12).

[0035] Carmovirus related primer Carmo-2 is based on the amino acid sequence V/PRT/VIQP (SEQ ID NO:13); the deduced nucleic acid sequence is: AA (AG) GTCGACCCG (AT) (AGCT) CC (AGCT) (AC) G (AGCT) GT (AGCT) AT (ACT) CA ACC. (SEQ ID NO:14)

[0036] The carrot mottle specific primers NC1 and NC2 are based on the amino acid sequences AKGFNAFQ (SEQ ID NO:15) and KYKVKG (SEQ ID NO:16) respectively; the deduced nucleic acid sequences being GC(AGCT)AA(AG)GG(AGCT)TT(CT)AATGC(AGCT)TT(CT)C (SEQ ID NO:17) for NC1 and (AG)CA(AGCT)CC(CT)TT(AGCT)AC(CT)TT(AG)TA(CT)TT (SEQ ID NO:18) for NC2.

[0037] The invention will now be further described with reference to the following non-limiting Figures and Examples. Other embodiments of the invention will occur to those skilled in the art in the light of these.

FIGURES

[0038]FIG. 1. shows the nucleotide sequence of CRLV cDNA (SEQ ID NO:9) which includes viral RdRp gene sequence.

[0039]FIG. 2 shows the predicted amino acid sequence of CRLV cDNA corresponding to the viral RdRp protein (SEQ ID NO:10)(a translation of part of CRLV sequence shown in FIG. 1).

[0040]FIG. 3a Shows a typical alignment of predicted amino acid sequence of CRLV cDNA shown in FIG. 2 with the predicted amino acid sequence of the RdRp proteins (replicase proteins) of Beet Western Yellows Virus (BWYV)(Accession no: P09507), Potato Leafroll Virus (PLRV)(Accession no:P17520), Pea Enation Mosaic Virus (PEMV)(Accession no:P29154) and Southern Bean Mosaic Virus (SBMV)(Accession no:P21405) The amino acid sequences used to design the degenerate RT -PCR primers are shown underlined.

[0041]FIG. 3b shows another typical alignment of the predicted amino acid sequence of CRLV cDNA shown in FIG. 2 with the predicted amino acid sequence of the RdRp proteins of Barley Yellow Dwarf Virus (isolate MAV-PS1)(BYDV-MAV-PS1)(Accession no:P29044), Barley Yellow Dwarf Virus (isolate P-PAV) (BYDV-P-PAV) (Accession no:p29045), Potato Leafroll Virus (PLRV-1) (Accession no:P17520), Potato Leafroll Virus (Wageningen Strain) (PLRV-WAG)(Accession no: P11623), Potato Leafroll Virus (Canadian isolate) (PLRV-CA) (Accession no:D00734), Beet Western Yellows Virus (Isolate FL-1) (BWYV-FL1) (Accession no: P09507), Beet Mild Yellowing Virus (BMYV), Barley Yellow Dwarf Virus (NY-RPV isolate) (BYDV-NY-RPV)(Accession no:L25299) and CRLV.

[0042]FIG. 4 shows the results of RT-PCR detection of CRLV in plant samples using primers CL1/CL2/CL3.

[0043]FIG. 5 shows the results of a CRLV RT-PCR sensitivity assay.

[0044]FIG. 6 shows the results of RT-PCR detection of CMoV in plant samples using primers NC1 and NC2.

EXAMPLES Example 1—Design of Degenerate Primers (CRLV)

[0045] Primers for generating and amplifying cDNAs from CRLV were designed using a previously unpublished partial sequence of CRLV and sequences from BWYV, PLRV, PEMV and SBMV corresponding to the RdRp gene in each of these luteoviruses (Accession nos: P09507, P17520, P29154 and P21405). The CRLV cDNA sequence is shown in FIG. 1 with a partial translation shown in FIG. 2. The translation was aligned with the known RdRp sequences of BWYV, PLRV, PEMV and SBMV using the GCG Wisconsin Package/PileUp software program.

[0046] The conserved regions on which the primers were based were chosen specifically as they comprise amino acids with a high degree of degeneracy. The conserved regions identified are shown underlined in FIGS. 3a and 3 b. The nucleic acid sequences of the primers are as follows: Primer CL1 based on Conserved Region FVKGEPH: 5′ TTY/GTN/AAR/GGN/GAR/CCN/CAY 3′ Primer CL2 based on Conserved Region GFKVEV: 5′ NAC/YTC/NAC/YTT/RAA/NCC 3′ (antisense and reverse orientation) Primer CL3 based on Conserved Region EDDMEV: CL3 = 5′ CK/NAC/YTC/CAT/RTC/RTC/YTC 3′ (antisense and reverse orientation)

Example 2—Nucleic Acid Extraction

[0047] Approximately 3 g of umbelliferous plant tissue from chervil plants was taken from each sample plant. Total plant RNA was prepared by using an RNeasy Plant Mini Kit (Qiagen). Unless otherwise stated, 3 g leaf material was taken from each plant and placed in a pestle and mortar. Before use, pestles and mortars were soaked in sodium hypochlorite solution, autoclaved and oven-baked to remove ribonucleases and possible traces of contaminating RNA. Liquid nitrogen was added to freeze the leaf material which was then ground to a fine powder. Carrot leaf powder (200 mg) was then added to 450 μl RLC buffer from the kit and total RNA was extracted using a Qi-shredder and an RNeasy column for each sample following manufacturer's instructions. Total RNA was eluted from the final column in 40 μl RNAse free water and stored at −20° C. This procedure gave typical RNA yields of 0.05 μg/ml.

Example 3—cDNA Synthesis and PCR Primer Annealing

[0048] 0.5 ml microfuge tubes were used throughout. 100 p mol (1 microlitre) RT-PCR primer (CL2) was added to 1 microlitre plant nucleic acid purified as above. Sterile water was added to 12 microlitres, in each tube.

[0049] The mixture was heated at 70° C. for 10 minutes (using a Gene E Techne Thermal cycler PCR block) and quick chilled on ice. 7 microlitres 100× first strand cDNA buffer and 1 microlitre (200 units) of Superscript™ RT enzyme was added. Samples were pulse spun and incubated at 37° C. for 1 hour using a PCR block. (First strand cDNA synthesis buffer stock was made up using 400 microlitres 5× first strand buffer (Gibco BRL), 200 microlitres 0.1 M DTT (Gibco, BRL) 100 microlitres mixed dNTP stock [10 mM each] dATP, dGTP, dCTP and dTTP made up from 100 mM stocks]). Finally, reverse transcriptase was denatured at 95° C. for 8 minutes.

Example 4—PCR Amplification of cDNA

[0050] For luteovirus detection, 5 μl cDNA was added to 95 μl PCR reaction mix [40 μl PCR primer CL1 at 100 pmol/ml, 40 μl PCR primer CL3 at 100 pmol/ml, 400 μl 10× RED Taq PCR Reaction Buffer (Sigma), 8 μl each dNTP (dATP/dGTP/dCTP/dTTP at 100 mM) for 0.2 mM final concentration, sterile water to 4 ml]. 2.5 μl (2.5 U) RED Taq™ DNA polymerase (Sigma) was then added to each tube, and samples were pulse spun and overlaid with a drop of mineral oil. PCR cycling was achieved using a Gene E Techne Thermal cycler PCR block as follows: 95° C. 1 minute denaturing, 45° C. 2 minutes annealing, 72° C. 4 minutes synthesis, for 40 cycles, 1 cycle of 72° C. 10 minute synthesis.

[0051] PCR products were analysed by gel electrophoresis. 15 μl PCR reaction and 5 μl gel loading dye were electrophoresed in 1.3% TAE agarose gels containing 0.3 μg/ml ethidium bromide, at 75 volts for 1 hour. DNA molecular weight markers (Bioline) were co-electrophoresed with the PCR products. As shown in FIG. 4 nucleic acid samples 1,4 and 8 tested CRLV positive.

Example 5—PCR Amplification of cDNA Using CL1 and CL2 as Primers

[0052] cDNA was amplified as in Example 4, but substituting CL2 for CL3. As with primers CL1/CL2/CL3, a number of nucleic acid samples were found to be positive for CRLV. An example is shown in FIG. 5.

Example 6—CRLV RT-PCR Sensitivity Assay

[0053] A comparison of the sensitivity of the test for CRLV using primers CL2/1/3 (as in Example 4) and CL2/1/2 (as in Example 5) was performed using carrot leaves of wild carrots obtained from a site in Oxfordshire. Nucleic acid was isolated and tested as described for the Chervil leaves above. The results are shown in FIG. 5.

[0054] Ten-fold serial dilutions were made of infected carrot leaf RNA with healthy carrot leaf RNA. In FIG. 5, H corresponds to 0.07 mg healthy carrot RNA used in cDNA synthesis with CRLV cDNA primer CL2. Lanes 1-4 correspond to undiluted 0.07 μg infected carrot RNA, ×10 diluted total infected carrot RNA, ×100 (700 pg infected carrot leaf RNA) and ×1000 diluted total infected carrot RNA. CRLV positive samples are shown in bold and underlined. PCR products obtained using three PCR primers are shown on the left and larger PCR products obtained using two PCR primers are shown on the right. A faint PCR band at ×100 dilution is obtained using three different PCR primers in RT-PCR. No PCR product is seen at ×100 dilution using two PCR primers. The results of a repeat dilution series experiment are shown on the lower half of the gel. RT-PCR using CL2 for cDNA synthesis and CL1/CL3 for PCR amplification of cDNA is shown as a more sensitive detection method than RT-PCR using CL2 for cDNA synthesis and CL1/CL2 for PCR amplification of cDNA.

Example 7—Design and Testing of of Degenerate Primers to Detect CMoV

[0055] The predicted polymerase amino acid sequence of the Australian isolate of CMoV (Gibbs, M. J., Cooper, J. I., and Waterhouse, P. M. (1996a).Virology 224:310-313; Gibbs, M. J., Zeigler, A., Robinson, D. J., Waterhouse, P. M., and Cooper, J. I. (1996b). Molecular Plant Pathology On-Line. [http://www.bspp.org.uk/mppol]1996/111gibbs) was aligned with the polymerase region of other carmo-like viruses and regions of amino acid conservation were used to select two degenerate RT-PCR primers (Carmo-2 and Carmo-6). This approach was used to PCR amplify and sequence a region of the polymerase gene of a UK isolate of CMoV. The predicted amino acid sequences of the Australian and UK isolates of CMoV were then aligned and two CMoV specific RT-PCR primers were designed (NC1 and NC2). Carmo-6 was used to prime cDNA synthesis and NC1 and NC2 were used for PCR amplification of cDNA.

[0056] The carmovirus related primer Carmo-6 is based on the amino acid sequence VCDDGNN; the deduced nucleic acid sequence is G(AC)(AC)CTGCAG(AGCT)AC(AG)CA(AG)TC(AG)TC(AGCT)CC(AG)TT(AG) TT. Carmovirus related primer Carmo-2 is based on the amino acid sequence V/PRT/VIQP; the deduced nucleic acid sequence AA(AG)GTCGACCCG(AT)(AGCT)CC(AGCT)(AC)G(AGCT)GT(AGCT)AT(ACT)CA ACC.

[0057] The carrot mottle specific NC1 and NC2 are based on the amino acid sequences AKGFNAFQ and KYKVKG respectively; the deduced nucleic acid sequences GC(AGCT)AA(AG)GG(AGCT)TT(CT)AATGC(AGCT)TT(CT)C for NC1 and (AG)CA(AGCT)CC(CT)TT(AGCT)AC(CT)TT(AG)TA(CT)TT for NC2.

[0058] The first strand cDNA was synthesised as in Example 3, substituting primer Carmo-6 in place of CL2. The cDNA sequence was amplified using the method as described in Example 4, substituting primers NC1 and NC2 in place of primers CL1 and CL3.

[0059] PCR products were analysed as described in Example 4. The results are shown in FIG. 6.

[0060] The major CMoV specific PCR product is predicted to be 258 bp long using NC1/NC2 primers in PCR following cDNA synthesis using Carmo-6 primer. CMoV positive samples are shown in bold and underlined (B). The white arrow marked indicates the position of the CMoV PCR product. A number of non-specific PCR bands larger than 258 bp were observed and included a PCR product of approximately 700 bp. A product of this size was seen in healthy controls and is assumed to be non-viral derived. Raising the primer annealing temperature from 45° C. to 47° C. did not remove background host-specific PCR products. Visualisation of degenerate RT-PCR products by gel analysis is therefore critical for determining CMoV positive samples.

1 41 1 7 PRT Carrot red leaf luteovirus 1 Phe Val Lys Gly Glu Pro His 1 5 2 21 DNA Artificial Sequence Description of Artificial Sequence Primer 2 ttygtnaarg gngarccnca y 21 3 6 PRT Carrot red leaf luteovirus 3 Gly Phe Lys Val Glu Val 1 5 4 18 DNA Artificial Sequence Description of Artificial Sequence Primer 4 nacytcnacy ttraancc 18 5 6 PRT Carrot red leaf luteovirus 5 Glu Asp Asp Met Glu Val 1 5 6 20 DNA Artificial Sequence Description of Artificial Sequence Primer 6 cknacytcca trtcrtcytc 20 7 6 PRT Carrot red leaf luteovirus 7 Lys Gly Glu Pro His Lys 1 5 8 19 DNA Artificial Sequence Description of Artificial Sequence Primer 8 aarggngarc cncayaarc 19 9 1100 DNA Carrot red leaf luteovirus misc_feature (21) n=a or g or c or t/u 9 cccgacaggt ttcccgactg naagcggcca gtgagcccaa cccaattaat gtgagttagc 60 tcactcatta ggcaccccag gctttacact ttatgcttcc ggctcgtatg ttgtgtggaa 120 ttgtgagcgg ataacaattt cacacaggaa acagctatga ccatgattac gaattcgagc 180 tcggtacccc gagaatgact tttgatcgac tacagaagat atcagaagtc aatttcagtg 240 atatgacacc ggaggagcta gttcaaaacg ggctctgcga tcctatcaga ctattcgtca 300 agggagagcc ccacaaacag agcaaactcg atgaaggccg ctaccgcctc ataatgagtg 360 tgtcattact agaccaattg gtagcccggg tcttgttcca gtctcagaac aagttagaaa 420 tcgcgctttg gagagcagta ccttcaaaac ccggatttgg cctttccaca gacaatcaaa 480 ttgaaaattt tgtagattgt ctagccaagc aagtggggga gacaccagaa gaggtcatag 540 ctaactggcc taaatactta atccccacag attgctcagg cttcgattgg tcagtttcag 600 actggctcct cgaagatgac atggaggtga gaaatcgctt gaccattgac tgcacccctc 660 ttttacggag gctgcgagca ggatggttga aatgcatttc caactctgtc ttgtgccttt 720 ctgatggcac cctgttagcc caaactatcc cccggggttc agaagagtgg atcgtataat 780 acgagttcct ctaattctag aatccgggtt atggcagcct atcattgtgg cgcctcttgg 840 gctatggcaa tgggcgatga tgctggaaag catagacacg gacctatccg gtataaaaat 900 ttgggtttaa agtcgaggtt cagcacaact ggaatttgct ctcacatatt taaggagaga 960 acctcgccat tccggtgaat atcaacaaat gtgtataact tatccacggt acatccggaa 1020 tgtggaacgc gagtctcagc actattaggc gtgctcgcat gatgaggtca gacgtacacg 1080 gactaagcca atcgttatgg 1100 10 18 PRT Carrot red leaf luteovirus SITE (7) Xaa is uncertain 10 Arg Gln Val Ser Arg Leu Xaa Ala Ala Ser Glu Pro Asn Pro Ile Asn 1 5 10 15 Val Ser 11 7 PRT Carrot mottle umbravirus 11 Val Cys Asp Asp Gly Asn Asn 1 5 12 30 DNA Artificial Sequence Description of Artificial Sequence Primer 12 gmmctgcagn acrcartcrt cnccrttrtt 30 13 6 PRT Carrot mottle umbravirus SITE (1) Xaa is Val or Pro 13 Xaa Arg Xaa Ile Gln Pro 1 5 14 31 DNA Artificial Sequence Description of Artificial Sequence Primer 14 aargtcgacc cgwnccnmgn gtnathcaac c 31 15 8 PRT Carrot mottle umbravirus 15 Ala Lys Gly Phe Asn Ala Phe Gln 1 5 16 6 PRT Carrot mottle umbravirus 16 Lys Tyr Lys Val Lys Gly 1 5 17 22 DNA Artificial Sequence Description of Artificial Sequence Primer 17 gcnaarggnt tyaatgcntt yc 22 18 21 DNA Artificial Sequence Description of Artificial Sequence Primer 18 rcanccyttn acyttrtayt t 21 19 20 PRT Carrot red leaf luteovirus 19 Ala Pro Gln Ala Leu His Phe Met Leu Pro Ala Arg Met Leu Cys Gly 1 5 10 15 Ile Val Ser Gly 20 20 214 PRT Carrot red leaf luteovirus 20 Gln Phe His Thr Gly Asn Ser Tyr Asp His Asp Tyr Glu Phe Glu Leu 1 5 10 15 Gly Thr Pro Arg Met Thr Phe Asp Arg Leu Gln Lys Ile Ser Glu Val 20 25 30 Asn Phe Ser Asp Met Thr Pro Glu Glu Leu Val Gln Asn Gly Leu Cys 35 40 45 Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His Lys Gln Ser Lys 50 55 60 Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val Ser Leu Leu Asp 65 70 75 80 Gln Leu Val Ala Arg Val Leu Phe Gln Ser Gln Asn Lys Leu Glu Ile 85 90 95 Ala Leu Trp Arg Ala Val Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr 100 105 110 Asp Asn Gln Ile Glu Asn Phe Val Asp Cys Leu Ala Lys Gln Val Gly 115 120 125 Glu Thr Pro Glu Glu Val Ile Ala Asn Trp Pro Lys Tyr Leu Ile Pro 130 135 140 Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ser Asp Trp Leu Leu Glu 145 150 155 160 Asp Asp Met Glu Val Arg Asn Arg Leu Thr Ile Asp Cys Thr Pro Leu 165 170 175 Leu Arg Arg Leu Arg Ala Gly Trp Leu Lys Cys Ile Ser Asn Ser Val 180 185 190 Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Thr Ile Pro Arg Gly 195 200 205 Ser Glu Glu Trp Ile Val 210 21 4 PRT Carrot red leaf luteovirus 21 Tyr Glu Phe Leu 1 22 19 PRT Carrot red leaf luteovirus 22 Asn Pro Gly Tyr Gly Ser Leu Ser Leu Trp Arg Leu Leu Gly Tyr Gly 1 5 10 15 Asn Gly Arg 23 4 PRT Carrot red leaf luteovirus 23 Cys Trp Lys Ala 1 24 6 PRT Carrot red leaf luteovirus 24 Thr Arg Thr Tyr Pro Val 1 5 25 18 PRT Carrot red leaf luteovirus 25 Lys Phe Gly Phe Lys Val Glu Val Gln His Asn Trp Asn Leu Leu Ser 1 5 10 15 His Ile 26 14 PRT Carrot red leaf luteovirus 26 Gly Glu Asn Leu Ala Ile Pro Val Asn Ile Asn Lys Cys Val 1 5 10 27 18 PRT Carrot red leaf luteovirus 27 Leu Ile His Gly Thr Ser Gly Met Trp Asn Ala Ser Leu Ser Thr Ile 1 5 10 15 Arg Arg 28 380 PRT Beet western yellows virus 28 Ala Gln Ser Ala Glu Ile Pro Ser Asp Ala Glu Arg Glu Arg Val Ile 1 5 10 15 Gln Lys Thr Ala Asp Val Tyr His Pro Cys Gln Thr Asn Gly Pro Ala 20 25 30 Ala Thr Arg Gly Gly Thr Leu Thr Trp Asn Asn Phe Met Ile Asp Phe 35 40 45 Lys Gln Ala Val Phe Ser Leu Glu Phe Asp Ala Gly Ile Glu Leu Pro 50 55 60 Tyr Ile Ala Tyr Gly Lys Pro Thr His Arg Gly Trp Val Glu Asp Gln 65 70 75 80 Lys Leu Leu Pro Ile Leu Ala Gln Leu Thr Phe Phe Arg Leu Gln Lys 85 90 95 Met Leu Glu Val Asn Phe Glu Asp Met Gly Pro Glu Glu Leu Val Arg 100 105 110 Asn Gly Leu Cys Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His 115 120 125 Lys Gln Ala Lys Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val 130 135 140 Ser Leu Val Asp Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn 145 150 155 160 Lys Arg Glu Ile Ala Leu Trp Arg Ala Ile Pro Ser Lys Pro Gly Phe 165 170 175 Gly Leu Ser Thr Asp Glu Gln Val Leu Asp Phe Val Glu Ser Leu Ala 180 185 190 Arg Gln Val Gly Thr Thr Thr Thr Glu Val Val Ala Asn Trp Lys Asn 195 200 205 Tyr Leu Thr Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala Asp 210 215 220 Trp Met Leu His Asp Asp Met Ile Val Arg Asn Arg Leu Thr Ile Asp 225 230 235 240 Leu Asn Pro Ala Thr Glu Arg Leu Arg Ser Cys Trp Leu Arg Cys Ile 245 250 255 Ser Asn Ser Val Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Ile 260 265 270 His Pro Gly Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser Asn 275 280 285 Ser Arg Ile Arg Val Met Ala Ala Phe His Thr Gly Ala Ile Trp Ala 290 295 300 Met Ala Met Gly Asp Asp Ala Leu Glu Ser Asn Pro Ala Asp Leu Ala 305 310 315 320 Ala Tyr Lys Lys Leu Gly Phe Lys Val Glu Val Ser Gly Gln Leu Glu 325 330 335 Phe Cys Ser His Ile Phe Arg Ala Pro Asp Leu Ala Leu Pro Val Asn 340 345 350 Glu Asn Lys Met Ile Tyr Lys Leu Ile Tyr Gly Tyr Asn Pro Gly Ser 355 360 365 Gly Asn Ala Glu Val Val Ser Asn Tyr Leu Ala Ala 370 375 380 29 381 PRT Potato leaf roll virus 29 Ala Glu Ser Ala Thr Ile Pro Gly Ala Glu Ala Arg Lys Arg Val Ile 1 5 10 15 Glu Lys Thr Val Glu Ala Tyr Arg Asn Cys Val Thr Asn Ala Pro Leu 20 25 30 Cys Ser Leu Lys Ser Lys Leu Asp Trp Ala Gly Phe Gln Gln Asp Ile 35 40 45 Arg Glu Ala Val Gln Ser Leu Glu Leu Asp Ala Gly Val Gly Ile Pro 50 55 60 Tyr Ile Ala Tyr Gly Leu Pro Ala His Arg Gly Trp Val Glu Asp His 65 70 75 80 Lys Leu Leu Pro Val Leu Thr Gln Leu Thr Phe Asp Arg Leu Gln Lys 85 90 95 Met Ser Glu Ala Ser Phe Glu Asp Met Ser Ala Glu Glu Leu Val Gln 100 105 110 Glu Gly Leu Cys Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His 115 120 125 Lys Gln Ser Lys Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val 130 135 140 Ser Leu Val Asp Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn 145 150 155 160 Lys Arg Glu Ile Ser Leu Trp Arg Ser Val Pro Ser Lys Pro Gly Phe 165 170 175 Gly Leu Ser Thr Asp Thr Gln Thr Ala Glu Phe Leu Glu Cys Leu Gln 180 185 190 Lys Val Ser Gly Ala Pro Ser Val Glu Glu Leu Cys Ala Asn His Lys 195 200 205 Glu Tyr Thr Arg Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala 210 215 220 Tyr Trp Met Leu Glu Asp Asp Met Glu Val Arg Asn Arg Leu Thr Phe 225 230 235 240 Asn Asn Thr Gln Leu Thr Lys Arg Leu Arg Ala Ala Trp Leu Lys Cys 245 250 255 Ile Gly Asn Ser Val Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln 260 265 270 Thr Val Pro Gly Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser 275 280 285 Asn Ser Arg Ile Arg Val Met Ala Ala Tyr His Cys Gly Ala Asp Trp 290 295 300 Ala Met Ala Met Gly Asp Asp Ala Leu Glu Ala Pro Asn Ser Asp Leu 305 310 315 320 Glu Glu Tyr Lys Thr Leu Gly Phe Lys Val Glu Val Gly Arg Glu Leu 325 330 335 Glu Phe Cys Ser His Ile Phe Arg Asn Pro Thr Leu Ala Val Pro Val 340 345 350 Asn Thr Asn Lys Met Leu Tyr Lys Leu Ile His Gly Tyr Asn Pro Glu 355 360 365 Cys Gly Asn Pro Glu Val Ile Gln Asn Tyr Leu Ala Ala 370 375 380 30 377 PRT Pea enation mosaic virus 30 Gln Thr Thr Ala Val Ile Pro Pro Lys Asp Val Arg Glu Asp Leu Ile 1 5 10 15 Lys Arg Thr Thr Glu Ala Tyr Arg Ser Thr Ala Leu Pro Ala Pro Met 20 25 30 Trp Ala His Asn Phe Asp Glu Ser His Met Arg Phe Glu Phe Trp Glu 35 40 45 Cys Val Arg Lys Leu Lys Gly Gln Ala Gly Ser Gly Val Pro Tyr Ala 50 55 60 Ala Phe Ser Gly Arg Lys Thr Asn Asp Lys Trp Val Phe Asp His Glu 65 70 75 80 Ser Thr Glu Asp Leu Trp Glu Thr Val Arg Asp Arg Leu Phe Arg Leu 85 90 95 Leu Asn Gln Asp Phe Ile Asp Pro Val Gln Ala Val Lys Asp Gly Leu 100 105 110 Val Asp Pro Ile Arg Leu Phe Val Lys Leu Glu Pro His Lys Met Glu 115 120 125 Lys Ile Arg Asn Lys Arg Tyr Arg Leu Ile Ala Ser Val Ser Ile Val 130 135 140 Asp Gln Leu Val Ala Arg Met Leu Phe Arg Asp Gln Asn Glu Glu Glu 145 150 155 160 Leu Leu Gln His Met Ala Ile Pro Ser Lys Pro Gly Leu Gly Phe Ser 165 170 175 Gln Asp His Gln Val Leu Ala Phe Thr Glu Ser Val Ala Ala Leu Ala 180 185 190 Gly Thr Ser Ala Gln Asp Leu Val Asp Asn Trp Ser Arg Tyr Leu Thr 195 200 205 Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Pro Met Trp Leu Leu 210 215 220 Glu Asp Asp Leu Ala Val Arg Asn Glu Leu Thr Leu Gly Leu Pro His 225 230 235 240 Gly Leu Arg Lys Met Arg Glu Thr Trp Leu Lys Cys Leu Gly Gln Ser 245 250 255 Val Phe Cys Leu Ser Asn Gly Leu Leu Leu Ala Gln Thr Ser Pro Gly 260 265 270 Ile Gln Lys Ser Gly Ser Phe Asn Thr Ser Ser Thr Asn Ser Arg Met 275 280 285 Arg Tyr Met Leu Ala Leu Tyr Ala Gly Ala Ser Trp Ala Val Thr Met 290 295 300 Gly Asp Asp Ala Leu Glu Ser Val Gly Ser Asp Leu Ser Gln Tyr Ala 305 310 315 320 Arg Leu Gly Ile Lys Cys Glu Arg Ala Glu Glu Phe Asp Phe Cys Ser 325 330 335 His Leu Phe Arg Ala Pro Asp Val Val Ile Pro Lys Asn Leu Glu Lys 340 345 350 Met Val Tyr Gly Leu Leu Ser Gly Thr Ser Pro Glu Ser Pro Leu Leu 355 360 365 Ala Asp Arg Phe Ser Trp Leu Ser Ala 370 375 31 11 PRT Carrot red leaf luteovirus 31 Leu Ile His Gly Thr Ser Gly Met Trp Asn Ala 1 5 10 32 379 PRT Southern bean mosaic virus 32 Gln Glu Ser Leu Phe Pro Pro Lys Pro Arg Ala Thr Ser Ser Lys Pro 1 5 10 15 Ile Thr Thr Ser Ser Pro Gly Thr Pro Gly Arg Ser Pro Leu Pro Val 20 25 30 Ser Gly Lys Glu Leu Gly Pro Ser Thr Gln Ser Ser Ser Lys Leu Ser 35 40 45 Arg Lys Gln Arg Arg Arg Arg Ser Thr Glu Lys Ala Ser Pro Gly Val 50 55 60 Pro Leu Ser Arg Leu Ala Thr Thr Asn Lys Asp Leu Met Ala Gln His 65 70 75 80 Met Gln Phe Val Ala Ala Cys Val Thr Gly Arg Val Pro Leu Leu Ala 85 90 95 Ser Phe Glu Asp Ile His Ala Leu Ser Pro Thr Glu Met Val Glu Met 100 105 110 Gly Leu Cys Asp Pro Val Arg Leu Phe Val Lys Gln Glu Pro His Pro 115 120 125 Ser Arg Lys Leu Lys Glu Gly Arg Tyr Arg Leu Ile Ser Ser Val Ser 130 135 140 Ile Val Asp Gln Leu Val Glu Arg Met Leu Phe Gly Ala Gln Asn Glu 145 150 155 160 Leu Glu Ile Ala Glu Trp Gln Ser Ile Pro Ser Lys Pro Gly Met Gly 165 170 175 Leu Ser Val Ile His Gln Ala Asp Ala Ile Phe Arg Asp Leu Arg Val 180 185 190 Lys His Thr Val Cys Pro Ala Ala Glu Ala Asp Ile Ser Gly Phe Asp 195 200 205 Trp Ser Val Gln Asp Trp Glu Leu Trp Ala Asp Val Glu Met Arg Ile 210 215 220 Val Leu Gly Ser Phe Pro Pro Met Met Ala Arg Ala Ala Arg Asn Arg 225 230 235 240 Phe Ser Cys Phe Met Asn Ser Val Leu Gln Leu Ser Asn Gly Gln Leu 245 250 255 Leu Gln Gln Glu Leu Pro Gly Ile Met Lys Ser Gly Ser Tyr Cys Thr 260 265 270 Ser Ser Thr Asn Ser Arg Ile Arg Cys Leu Met Ala Glu Leu Ile Gly 275 280 285 Ser Pro Trp Cys Ile Ala Met Gly Asp Asp Ser Val Glu Gly Phe Val 290 295 300 Glu Gly Ala Arg Glu Lys Tyr Ala Gly Leu Gly His Leu Cys Lys Asp 305 310 315 320 Tyr Lys Pro Cys Ala Thr Thr Pro Thr Gly Gln Leu Tyr Ala Val Glu 325 330 335 Phe Cys Ser His Val Ile Lys Arg Asn Lys Ala Phe Leu Thr Ser Trp 340 345 350 Pro Lys Thr Leu Tyr Arg Phe Leu Ser Thr Pro Arg Glu Thr Leu Glu 355 360 365 Asp Leu Glu Arg Glu Leu Ala Ser Ser Pro Met 370 375 33 562 PRT Beet mild yellowing virus 33 Cys Ala Asn Arg Val Leu Pro Asp Ala Gly Gly Arg Lys Gly Gly Ser 1 5 10 15 Asp Arg Arg Asn Asn Arg Lys Arg Lys His Pro Arg Glu Ile Arg Arg 20 25 30 Lys Trp Lys Lys Pro Ser Cys Cys Ser Phe Tyr Thr Ala Gly Thr Leu 35 40 45 Gly Glu Asn Cys Thr Ala Ser His Val His Cys Thr Ser Lys Glu Glu 50 55 60 Tyr Asp Glu Trp Pro Arg Cys Trp Cys Gln Ile Ala Gly His Asp Cys 65 70 75 80 His Tyr Arg Ser Asn Leu Arg Asp Lys Glu Gly Ser Asp Arg Gln Asn 85 90 95 Gly Phe Glu Ile Asp Arg Glu Thr Ser Gly Arg Asp Thr Ile Val Asp 100 105 110 Gly His Glu Glu Ala Pro Leu Lys Arg Ala Glu Lys Ile Gln Glu Gln 115 120 125 Ala Lys Gln Phe Gly Arg Phe Phe Lys Thr Gln Tyr His Trp Glu Arg 130 135 140 Ala Ala Glu Val Cys Pro Gly Phe Ile Lys Val Gly Glu Leu Pro Lys 145 150 155 160 Phe Tyr Phe Ser Lys Gln Lys Gly Cys Ser Asp Trp Gly Thr Lys Leu 165 170 175 Thr Ser Leu His Pro Glu Leu Glu Glu Lys Thr Arg Gly Phe Gly Trp 180 185 190 Pro Lys Phe Gly Pro Ala Ala Glu Leu Lys Ser Leu Arg Leu Gln Ala 195 200 205 Ala Arg Trp Leu Glu Arg Ala Glu Gln Val Lys Ile Pro Ser Thr Glu 210 215 220 Glu Arg Glu Arg Val Val Arg Lys Cys Val Glu Ala Phe Ser Pro Thr 225 230 235 240 Gln Thr Arg Gly Pro Met Ala Thr Arg Gly Asn Lys Leu Ser Trp Asn 245 250 255 Asn Phe Leu Glu Asp Phe Lys Thr Ala Val Phe Ser Leu Glu Leu Glu 260 265 270 Ala Gly Val Gly Val Pro Tyr Val Ala Tyr Gly Arg Arg Thr His Arg 275 280 285 Gly Trp Ile Glu Asp Pro Asp Leu Leu Pro Val Leu Ala Arg Phe Thr 290 295 300 Phe Asp Arg Leu Gln Lys Leu Ser Glu Val Lys Phe Glu His Met Ser 305 310 315 320 Pro Glu Gln Leu Val Gln Glu Gly Leu Cys Asp Pro Ile Arg Leu Phe 325 330 335 Val Lys Gly Glu Pro His Lys Gln Ser Lys Leu Asp Glu Gly Arg Tyr 340 345 350 Arg Leu Ile Met Ser Val Ser Leu Val Asp Gln Leu Val Ala Arg Val 355 360 365 Leu Phe Gln Asn Gln Asn Lys Arg Glu Ile Ala Leu Trp Arg Ala Ile 370 375 380 Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr Asp Gly Gln Val Val Asp 385 390 395 400 Phe Met Gln Ala Leu Ser Ala Gln Val Gly Val Asn Thr Ala Glu Leu 405 410 415 Leu Gln Asn Trp Lys Ser His Leu Ile Pro Thr Asp Cys Ser Gly Phe 420 425 430 Asp Trp Ser Val Ser Asp Trp Leu Leu Glu Asp Glu Met Glu Val Arg 435 440 445 Asn Arg Leu Thr Leu Asp Ile Asn Asp Leu Thr Arg Arg Leu Arg Ala 450 455 460 Gly Trp Leu Lys Cys Leu Ala Asn Ser Val Leu Cys Leu Ser Asp Gly 465 470 475 480 Thr Leu Leu Ser Gln Gln Val Pro Gly Val Gln Lys Ser Gly Ser Tyr 485 490 495 Asn Thr Ser Ser Ser Asn Ser Arg Ile Arg Val Met Ala Ala Tyr His 500 505 510 Ser Gly Ala Ser Trp Ala Ile Ala Met Gly Asp Asp Ala Leu Glu Ser 515 520 525 Val Asp Ala Asp Leu Ser Arg Tyr Ser Ser Leu Gly Phe Lys Val Glu 530 535 540 Val Ser Ser Gln Leu Glu Phe Cys Ser His Ile Phe Glu Glu Glu Asn 545 550 555 560 Leu Ala 34 565 PRT CABV 34 Ile Arg Val Gly Lys Arg Ala Gly Gly Gly Asp Arg Arg Asn Asn Arg 1 5 10 15 Thr Arg Cys His Pro Arg Gly Trp Ala His Lys Trp Arg Arg Thr Val 20 25 30 Ser Cys Cys Ser Leu Phe Pro Glu Gly Gln His Val His Phe Thr Thr 35 40 45 Ser Glu Gln Thr Gly Lys Pro His Cys Phe Cys Ser Ile Pro His His 50 55 60 Gln Cys Phe Tyr Arg His Phe Val Gly Asn Gln Thr Gly Asn Asn Gly 65 70 75 80 Gln Asn Arg Cys Pro Phe Asn Arg Glu Ala Ser Gly Pro Ser Leu Gly 85 90 95 Gly Gln Ser His Glu Glu Thr Glu Val Ala Phe Thr Gln Thr Arg Gln 100 105 110 Glu Lys Phe Glu Glu Leu Ala Thr Asp Phe Ser Ser Phe Phe Asp Ala 115 120 125 Gln Tyr Thr Trp Glu Gln Gly Gly Glu Glu Ala Pro Gly Phe Asp Lys 130 135 140 Val Gly Ser Leu Pro Gln Phe Tyr His Ala Lys Gln Lys Lys Ser Ser 145 150 155 160 Asn Trp Gly Asp Lys Ile Cys Phe Thr Lys Gln His Pro Glu Met Gly 165 170 175 Asp Leu Thr Lys Gly Phe Gly Trp Pro Gln Phe Gly Ala Lys Ala Glu 180 185 190 Leu Lys Ser Leu Arg Leu Gln Ala Ala Arg Trp Leu Glu Arg Ala Gln 195 200 205 Ser Val Lys Ile Pro Ser Ser Glu Glu Arg Glu His Val Ile Glu Arg 210 215 220 Cys Cys Arg Ala Phe Thr Tyr Gln Ala Ala Lys Thr Asn Gly Pro Met 225 230 235 240 Ala Thr Arg Gly Asp Arg Leu Ser Trp Asp Asn Phe Leu Gln Asp Phe 245 250 255 Lys Gln Ala Val Leu Ser Leu Glu Met Asp Ala Gly Ile Gly Val Pro 260 265 270 Tyr Ile Ala Tyr Gly Lys Pro Thr His Arg Gly Trp Val Glu Asp Lys 275 280 285 Lys Phe Thr Leu Leu Pro Ile Leu Ala Arg Leu Thr Phe Asn Arg Leu 290 295 300 Gln Lys Met Leu Glu Val Arg Tyr Val Asp Leu Ser Pro Ala Glu Leu 305 310 315 320 Val Arg Arg Gly Leu Cys Asp Pro Ile Arg Val Phe Val Lys Gly Glu 325 330 335 Pro His Lys Gln Ala Lys Leu Asp Glu Gly Arg Tyr Arg Leu Phe Thr 340 345 350 Ile Met Ser Val Ser Leu Val Asp Gln Leu Val Ala Arg Val Leu Phe 355 360 365 Gln Asn Gln Asn Lys Arg Glu Ile Ala Leu Trp Arg Val Val Pro Ser 370 375 380 Lys Pro Gly Phe Gly Leu Ser Thr Asp Glu Gln Val Ala Glu Phe Met 385 390 395 400 Gln Ile Leu Ser Ala Gln Val Gly Leu Thr Pro Phe Thr Ser Asp Leu 405 410 415 Ile Thr Glu Trp Arg Ala His Met Ile Ala Thr Asp Cys Ser Gly Phe 420 425 430 Asp Trp Ser Val Ser Asp Trp Leu Leu Glu Asp Asp Met Glu Val Arg 435 440 445 Asn Arg Leu Thr Leu Asp Leu Asn Glu Thr Thr Arg Arg Leu Arg Ser 450 455 460 Trp Leu Tyr Cys Ile Ser Asn Ser Phe Thr Val Leu Cys Leu Ser Asp 465 470 475 480 Gly Thr Leu Leu Ala Gln Arg Val Pro Gly Val Gln Lys Ser Gly Ser 485 490 495 Tyr Asn Thr Ser Ser Ser Asn Ser Arg Ile Arg Val Met Ala Ala Tyr 500 505 510 His Cys Gly Ala Glu Trp Ala Met Ala Met Gly Asp Asp Ala Leu Glu 515 520 525 Ser Ala Cys Ser Asn Phe Thr Leu Glu Arg Tyr Lys Ser Leu Gly Phe 530 535 540 Lys Val Glu Glu Ser Ser Lys Leu Glu Phe Cys Ser His Ile Phe Glu 545 550 555 560 Lys Glu Asp Leu Ala 565 35 520 PRT Beet western yellows virus 35 Arg His Phe Leu Arg Lys Gln Arg Arg Ser Val Gly Lys Arg Ser Ala 1 5 10 15 Arg His Arg Pro Arg Asn Lys Arg Arg Arg Gln Leu His Pro Lys Asp 20 25 30 Lys Gln Arg Arg Trp Glu Arg Asp Asp Gly Glu Asn Asn Leu Ile Ser 35 40 45 Ser Gly Lys Asp Lys Ser Arg Glu His Arg Glu Glu Ser Asp Arg Gly 50 55 60 Asp Leu Arg Glu Ser Asp Glu Asn Ser Glu Ile Pro Pro Gln Lys Ser 65 70 75 80 Pro Lys Glu Thr Ala Gly Glu Phe Glu Arg Tyr Phe Ser Ser Leu Tyr 85 90 95 Asn Trp Glu Val Pro Thr Ser Pro Arg Glu Val Pro Gly Phe Arg His 100 105 110 Cys Gly Lys Leu Pro Gln Tyr Tyr His Pro Lys Gln Lys Glu Glu Ser 115 120 125 Ser Trp Gly Lys Thr Leu Val Gly Asn His Pro Ala Leu Gly Glu Lys 130 135 140 Thr Ser Gly Phe Gly Trp Pro Lys Phe Gly Pro Glu Ala Glu Leu Lys 145 150 155 160 Ser Leu Arg Leu Gln Ala Ser Arg Trp Leu Glu Arg Ala Gln Ser Ala 165 170 175 Glu Ile Pro Ser Asp Ala Glu Arg Glu Arg Val Ile Gln Lys Thr Ala 180 185 190 Asp Val Tyr His Pro Cys Gln Thr Asn Gly Pro Ala Ala Thr Arg Gly 195 200 205 Gly Thr Leu Thr Trp Asn Asn Phe Met Ile Asp Phe Lys Gln Ala Val 210 215 220 Phe Ser Leu Glu Phe Asp Ala Gly Ile Glu Leu Pro Tyr Ile Ala Tyr 225 230 235 240 Gly Lys Pro Thr His Arg Gly Trp Val Glu Asp Gln Lys Leu Leu Pro 245 250 255 Ile Leu Ala Gln Leu Thr Phe Phe Arg Leu Gln Lys Met Leu Glu Val 260 265 270 Asn Phe Glu Asp Met Gly Pro Glu Glu Leu Val Arg Asn Gly Leu Cys 275 280 285 Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His Lys Gln Ala Lys 290 295 300 Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val Ser Leu Val Asp 305 310 315 320 Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn Lys Arg Glu Ile 325 330 335 Ala Leu Trp Arg Ala Ile Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr 340 345 350 Asp Glu Gln Val Leu Asp Phe Val Glu Ser Leu Ala Arg Gln Val Gly 355 360 365 Thr Thr Thr Thr Glu Val Val Ala Asn Trp Lys Asn Tyr Leu Thr Pro 370 375 380 Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala Asp Trp Met Leu His 385 390 395 400 Asp Asp Met Ile Val Arg Asn Arg Leu Thr Ile Asp Leu Asn Pro Ala 405 410 415 Thr Glu Arg Leu Arg Ser Cys Trp Leu Arg Cys Ile Ser Asn Ser Val 420 425 430 Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Ile His Pro Gly Val 435 440 445 Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser Asn Ser Arg Ile Arg 450 455 460 Val Met Ala Ala Phe His Thr Gly Ala Ile Trp Ala Met Ala Met Gly 465 470 475 480 Asp Asp Ala Leu Glu Ser Asn Pro Ala Asp Leu Ala Ala Tyr Lys Lys 485 490 495 Leu Gly Phe Lys Val Glu Val Ser Gly Gln Leu Glu Phe Cys Ser His 500 505 510 Ile Phe Arg Ala Pro Asp Leu Ala 515 520 36 553 PRT Potato leaf roll virus 36 Gly Leu Arg Ser Gly Glu Arg Gly Cys Asn Lys Cys Ala Arg Arg Glu 1 5 10 15 Asn Cys Ser Asn Lys Leu Ser Arg Glu Asp Cys Ser Ile Asn Phe Ser 20 25 30 Arg Glu Asn Cys Ser Asn Lys Gln Ala Phe Lys Trp Ala Ser Gly Thr 35 40 45 Val Arg Gln Asn Lys Arg Gln Leu Arg His Pro Arg Arg Arg Tyr Lys 50 55 60 Arg Thr Thr Asn Gly Gln Asn Gly Arg Thr Asp His His Ser Tyr Gly 65 70 75 80 Gly Glu Asn Gln Ser Leu Gly Asp Arg Gly Glu Asp Ser Glu Gln Gly 85 90 95 Val Ser Glu Ser Pro Ala Glu Ala Gln Thr Lys Glu Ala Arg Lys Ala 100 105 110 Trp Arg Glu Glu Gln Ala Lys Gln Phe Thr Ser Tyr Phe Asn Ala Ile 115 120 125 Tyr Lys Trp Gly Ala Gln Glu Gly Gly Cys Pro Pro Gly Phe Arg Lys 130 135 140 Cys Gly His Ile Pro Arg Tyr Tyr His Pro Arg Thr Arg Gly Glu Thr 145 150 155 160 Gln Trp Gly Gln Lys Leu Cys Gln Val His Pro Glu Leu Ala Asp Lys 165 170 175 Thr Ala Gly Phe Gly Trp Pro Lys Ala Gly Ser Glu Ala Glu Leu Gln 180 185 190 Ser Leu Asn Leu Gln Ala Ala Arg Trp Leu Gln Arg Ala Glu Ser Ala 195 200 205 Thr Ile Pro Gly Ala Glu Ala Arg Lys Arg Val Ile Glu Lys Thr Val 210 215 220 Glu Ala Tyr Arg Asn Cys Val Thr Asn Ala Pro Leu Cys Ser Leu Lys 225 230 235 240 Ser Lys Leu Asp Trp Ala Gly Phe Gln Gln Asp Ile Arg Glu Ala Val 245 250 255 Gln Ser Leu Glu Leu Asp Ala Gly Val Gly Ile Pro Tyr Ile Ala Tyr 260 265 270 Gly Leu Pro Ala His Arg Gly Trp Val Glu Asp His Lys Leu Leu Pro 275 280 285 Val Leu Thr Gln Leu Thr Phe Asp Arg Leu Gln Lys Met Ser Glu Ala 290 295 300 Ser Phe Glu Asp Met Ser Ala Glu Glu Leu Val Gln Glu Gly Leu Cys 305 310 315 320 Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His Lys Gln Ser Lys 325 330 335 Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val Ser Leu Val Asp 340 345 350 Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn Lys Arg Glu Ile 355 360 365 Ser Leu Trp Arg Ser Val Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr 370 375 380 Asp Thr Gln Thr Ala Glu Phe Leu Glu Cys Leu Gln Lys Val Ser Gly 385 390 395 400 Ala Pro Ser Val Glu Glu Leu Cys Ala Asn His Lys Glu Tyr Thr Arg 405 410 415 Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala Tyr Trp Met Leu 420 425 430 Glu Asp Asp Met Glu Val Arg Asn Arg Leu Thr Phe Asn Asn Thr Gln 435 440 445 Leu Thr Lys Arg Leu Arg Ala Ala Trp Leu Lys Cys Ile Gly Asn Ser 450 455 460 Val Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Thr Val Pro Gly 465 470 475 480 Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser Asn Ser Arg Ile 485 490 495 Arg Val Met Ala Ala Tyr His Cys Gly Ala Asp Trp Ala Met Ala Met 500 505 510 Gly Asp Asp Ala Leu Glu Ala Pro Asn Ser Asp Leu Glu Glu Tyr Lys 515 520 525 Thr Leu Gly Phe Lys Val Glu Val Gly Arg Glu Leu Glu Phe Cys Ser 530 535 540 His Ile Phe Arg Asn Pro Thr Leu Ala 545 550 37 553 PRT Potato leaf roll virus 37 Gly Leu Arg Ser Gly Glu Arg Gly Cys Asn Lys Cys Ala Arg Arg Glu 1 5 10 15 Asn Cys Ser Asn Lys Leu Ser Arg Glu Asp Cys Ser Ile Asn Phe Ser 20 25 30 Arg Glu Asn Cys Ser Asn Lys Gln Ala Phe Lys Trp Ala Ser Gly Thr 35 40 45 Ala Arg Gln Asn Lys Arg Gln Leu Arg His Pro Arg Arg Arg Tyr Lys 50 55 60 Arg Thr Thr Asn Gly Gln Asn Gly Arg Thr Asp His His Ser Tyr Gly 65 70 75 80 Gly Glu Asn Gln Ser Leu Gly Asp Arg Gly Glu Asp Ser Glu Gln Gly 85 90 95 Val Ser Glu Ser Pro Ala Glu Ala Gln Thr Lys Gln Thr Arg Lys Thr 100 105 110 Trp Arg Glu Glu Gln Ala Lys Gln Phe Thr Ser Tyr Phe Asp Ala Ile 115 120 125 Tyr Lys Trp Gly Ala Gln Glu Glu Gly Cys Pro Pro Gly Phe Arg Lys 130 135 140 Cys Gly Asn Ile Pro Gly Tyr Tyr His Pro Arg Thr Lys Gly Glu Thr 145 150 155 160 Lys Trp Gly Gln Lys Leu Cys Gln Val His Pro Glu Leu Ala Asp Lys 165 170 175 Thr Ala Gly Phe Gly Trp Pro Lys Ala Gly Phe Glu Ala Glu Leu Gln 180 185 190 Ser Leu Asn Leu Gln Ala Ala Arg Trp Leu Gln Arg Ala Glu Ser Ala 195 200 205 Thr Ile Pro Gly Ala Glu Ala Arg Lys Arg Val Ile Glu Lys Thr Val 210 215 220 Glu Ala Tyr Arg Asn Cys Ile Thr Asn Ala Pro Leu Cys Ser Leu Lys 225 230 235 240 Ser Lys Leu Asp Trp Ala Gly Phe Gln Gln Asp Ile Arg Glu Ala Val 245 250 255 Gln Ser Leu Glu Leu Asp Ala Gly Val Gly Ile Pro Tyr Ile Ala Tyr 260 265 270 Gly Leu Pro Thr His Arg Gly Trp Val Glu Asp His Lys Leu Leu Pro 275 280 285 Val Leu Thr Gln Leu Thr Phe Asp Arg Leu Gln Lys Met Ser Glu Ala 290 295 300 Ser Phe Glu Asp Met Ser Ala Glu Glu Leu Val Gln Glu Gly Leu Cys 305 310 315 320 Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His Lys Gln Ser Lys 325 330 335 Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val Ser Leu Val Asp 340 345 350 Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn Lys Arg Glu Ile 355 360 365 Ser Leu Trp Arg Ser Val Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr 370 375 380 Asp Thr Gln Thr Ala Glu Phe Leu Glu Cys Leu Gln Lys Val Ser Gly 385 390 395 400 Ala Pro Ser Val Glu Glu Leu Cys Ala Asn His Lys Glu His Thr Arg 405 410 415 Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala Tyr Trp Met Leu 420 425 430 Glu Asp Asp Met Glu Val Arg Asn Arg Leu Thr Phe Asn Asn Thr Gln 435 440 445 Leu Thr Glu Arg Leu Arg Ala Ala Trp Leu Lys Cys Ile Gly Asn Ser 450 455 460 Val Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Thr Val Pro Gly 465 470 475 480 Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser Asn Ser Arg Ile 485 490 495 Arg Val Met Ala Ala Tyr His Cys Gly Ala Asp Trp Ala Met Ala Met 500 505 510 Gly Asp Asp Ala Leu Glu Ala Pro Asn Ser Asp Leu Glu Glu Tyr Lys 515 520 525 Thr Leu Gly Phe Lys Val Glu Val Gly Arg Glu Leu Glu Phe Cys Ser 530 535 540 His Ile Phe Arg Asn Pro Thr Leu Ala 545 550 38 553 PRT Potato leaf roll virus 38 Gly Leu Arg Ser Gly Glu Arg Gly Cys Asn Lys Cys Ala Arg Arg Glu 1 5 10 15 Asn Cys Ser Asn Lys Leu Ser Arg Glu Asp Cys Ser Ile Asn Phe Ser 20 25 30 Arg Glu Asn Cys Ser Asn Lys Gln Ala Phe Lys Trp Ala Ser Gly Thr 35 40 45 Val Arg Gln Asn Lys Arg Gln Leu Arg His Pro Arg Arg Arg Tyr Lys 50 55 60 Arg Thr Thr Asn Glu Gln Asn Gly Arg Thr Asp His His Ser Tyr Gly 65 70 75 80 Gly Glu Asn Gln Ser Leu Gly Asp Arg Gly Glu Asp Arg Val Gln Gly 85 90 95 Val Ser Glu Ser Pro Ala Glu Ala Gln Thr Lys Glu Thr Arg Lys Ala 100 105 110 Trp Arg Glu Glu Gln Ala Lys Gln Phe Thr Ser Tyr Phe Asp Ala Ile 115 120 125 Tyr Lys Trp Gly Ala Gln Glu Gly Gly Cys Pro Pro Gly Phe Arg Lys 130 135 140 Cys Gly Asn Ile Pro Gly Tyr Tyr His Pro Arg Thr Arg Gly Gln Thr 145 150 155 160 Lys Trp Gly Gln Lys Leu Cys Gln Val His Pro Glu Leu Ala Glu Lys 165 170 175 Thr Ala Gly Phe Gly Trp Pro Lys Ala Gly Ser Glu Ala Glu Leu Gln 180 185 190 Ser Leu Asn Leu Gln Ala Ala Arg Trp Leu Gln Arg Ala Glu Ser Ala 195 200 205 Thr Ile Pro Gly Ala Glu Ala Arg Lys Arg Val Ile Glu Lys Thr Val 210 215 220 Glu Thr Tyr Arg Asn Cys Val Thr Asn Ala Pro Leu Cys Ser Leu Lys 225 230 235 240 Ser Lys Leu Asp Trp Ala Gly Phe Gln Gln Asp Ile Arg Glu Ala Val 245 250 255 Gln Ser Leu Glu Leu Asp Ala Gly Val Gly Ile Pro Tyr Ile Ala Tyr 260 265 270 Gly Leu Pro Thr His Arg Gly Trp Val Glu Asp His Lys Leu Leu Pro 275 280 285 Val Leu Thr Gln Leu Thr Phe Asp Arg Leu Gln Lys Met Ser Glu Ala 290 295 300 Ser Phe Glu Asp Met Ser Ala Glu Glu Leu Val Gln Glu Gly Leu Cys 305 310 315 320 Asp Pro Ile Arg Leu Phe Val Lys Gly Glu Pro His Lys Gln Ser Lys 325 330 335 Leu Asp Glu Gly Arg Tyr Leu Leu Ile Met Ser Val Ser Leu Val Asp 340 345 350 Gln Leu Val Ala Arg Val Leu Phe Gln Asn Gln Asn Lys Arg Glu Ile 355 360 365 Ser Leu Trp Arg Ser Val Pro Ser Lys Pro Gly Phe Gly Leu Ser Thr 370 375 380 Asp Thr Gln Thr Ala Glu Phe Leu Glu Cys Leu Gln Lys Val Tyr Gly 385 390 395 400 Ala Pro Ser Val Glu Glu Leu Cys Ala Asn His Lys Glu Tyr Thr Arg 405 410 415 Ser Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ala Tyr Trp Met Leu 420 425 430 Glu Asp Asp Met Glu Val Arg Asn Arg Leu Thr Phe Asn Asn Thr Gln 435 440 445 Leu Thr Lys Arg Leu Arg Ala Ala Trp Leu Lys Cys Ile Gly Asn Ser 450 455 460 Val Leu Cys Leu Ser Asp Gly Thr Leu Leu Ala Gln Thr Val Pro Gly 465 470 475 480 Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Ser Asn Ser Arg Ile 485 490 495 Arg Val Met Ala Ala Tyr His Cys Gly Ala Asp Trp Ala Met Ala Met 500 505 510 Gly Asp Asp Ala Leu Glu Ala Pro Asn Ser Asp Leu Glu Glu Tyr Lys 515 520 525 Thr Leu Gly Phe Lys Val Glu Val Gly Arg Glu Leu Glu Phe Cys Ser 530 535 540 His Ile Phe Arg Asn Pro Thr Leu Ala 545 550 39 570 PRT Barley yellow dwarf virus 39 Lys Arg His Gln Leu Gly Arg His Arg Arg Arg Val Gly Lys Arg Glu 1 5 10 15 Gly Gly Gly Val Arg Arg Asn Lys Arg Ser Ser Gly Lys Gln Gly Tyr 20 25 30 Arg His Pro Arg Arg Arg Gln Val Pro Lys Glu Asp Cys Cys Ser Phe 35 40 45 Phe Ala Glu Gly Thr Ala Ala Pro Arg Cys Ile Pro Thr His Gln His 50 55 60 Asn Trp Glu Gln Asn Ser Tyr Met Ser Asn Cys Tyr Cys Asn Ile Pro 65 70 75 80 Gly His Gly Cys Ala Tyr Trp Thr Ile Ala Ala Arg Asn His Glu Gln 85 90 95 His Asn Glu Ser Ala Gly Pro Glu Asp Arg Tyr Val Glu Asp Arg Glu 100 105 110 Ile Asp Ser Gly Pro Ser Arg Glu Ser Ser Ser Glu Glu Thr Thr Arg 115 120 125 Gln Ala Trp Leu Lys Glu Thr Ala Arg Ser Trp Gln Lys Phe Phe Ala 130 135 140 Asp Ile Tyr Thr Trp Asp Val Ser Thr Ser Lys Gln Glu Val Pro Gly 145 150 155 160 Phe Glu Gln Val Gly Lys Phe Ser Pro Gln Tyr Tyr Pro Arg Pro Arg 165 170 175 Ser Glu Ser Glu Trp Gly Arg Lys Leu Cys Ala Glu His Pro Leu Leu 180 185 190 Gly Glu Lys Thr Ala Gly Phe Gly Trp Pro Gln Val Gly Ala Ser Ala 195 200 205 Glu Leu Thr Ser Leu Arg Leu Gln Ala Ala Arg Trp Leu Glu Arg Ser 210 215 220 Glu Ser Ala Lys Ile Pro Ser Asp Ala Ala Arg Glu Asn Val Ile Asn 225 230 235 240 Arg Thr Val Gln Ala Tyr Ser Asn Cys Lys Thr Asn Thr Pro Arg Cys 245 250 255 Thr Arg Gly Glu Leu Ser Trp Glu Thr Phe Lys Ile Asp Phe Leu Glu 260 265 270 Ala Ile Lys Ser Leu Gln Leu Asp Ala Gly Val Gly Leu Pro Met Ile 275 280 285 Thr Ala Gly Leu Pro Thr His Arg Gly Trp Val Glu Asp Pro Asp Arg 290 295 300 Leu Pro Val Leu Ala Gln Leu Thr Phe Asp Arg Leu Leu Thr Met Ser 305 310 315 320 Lys Ala Ser Leu Glu Ala Arg Ser Pro Glu Gln Leu Val Lys Glu Asn 325 330 335 Leu Cys Asp Pro Ile Arg Leu Phe Val Lys Gln Glu Pro His Lys Gln 340 345 350 Ser Lys Leu Asp Glu Gly Arg Tyr Arg Leu Ile Met Ser Val Ser Leu 355 360 365 Ile Asp Gln Leu Val Ala Arg Val Leu Phe Gln Arg Gln Asn Lys Ser 370 375 380 Glu Ile Ala Leu Trp Ser Ala Ile Pro Ser Lys Pro Gly Phe Gly Leu 385 390 395 400 Ser Thr Glu Asp Gln Val Ser Lys Phe Met Asp Val Leu Ala Gly Asn 405 410 415 Val Gly Ala Ser Pro Glu Glu Val Cys Asp Asn Trp Arg Asp Leu Leu 420 425 430 Val Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Ser Asp Trp Met 435 440 445 Leu Ala Asp Asp Met Glu Val Arg Asn Arg Leu Thr Ile Asp Cys Asn 450 455 460 Glu Leu Thr Arg His Leu Arg Ala Val Trp Leu Gln Gly Ile Ser Asn 465 470 475 480 Ser Val Leu Cys Leu Ser Asp Gly Thr Met Leu Ala Gln Arg Val Pro 485 490 495 Gly Val Gln Lys Ser Gly Ser Tyr Asn Thr Ser Ser Thr Asn Ser Arg 500 505 510 Val Arg Val Met Ala Ala Tyr His Cys Gly Ala Ser Trp Ala Ile Ala 515 520 525 Met Gly Asp Asp Ala Leu Glu Ala Pro Asp Thr Asp Leu Ser Lys Tyr 530 535 540 Lys Asp Leu Gly Phe Lys Val Glu Val Ser Gly Glu Leu Glu Phe Cys 545 550 555 560 Ser Arg Ile Phe Lys Thr Pro Asn Leu Ala 565 570 40 5 PRT Carrot red leaf luteovirus 40 Gly Glu Asn Leu Ala 1 5 41 537 PRT Pea enation mosaic virus 41 Met Ser Pro Gln Thr Leu Gly Lys Arg Ile Ile Pro Val Asp Ser Gly 1 5 10 15 Glu Thr Lys Ser Ser Glu Asp Pro Leu Pro Lys Gly Arg Gly Val Ser 20 25 30 Ser Thr Pro Ser Arg Ser Lys Ser Arg Lys Gly Lys Ala Cys Pro Ser 35 40 45 Phe Arg Asn Asp Ala Gly Thr Glu Glu Ser Arg Gln Pro Gln Glu Glu 50 55 60 Lys Gly Gln Ser Cys Gln Glu Asp Ser Leu Asn Ser Thr Gln Glu Ile 65 70 75 80 Gln Gly Gln Ser Thr His Phe Val Pro Ser Ser Gly Thr Gly Arg Lys 85 90 95 Ser Cys Glu Ser Ser Pro His Arg Pro Thr Thr Lys Ile Thr Ser Ile 100 105 110 Phe Glu Asp Phe Tyr Arg Trp Lys Glu Pro Arg Glu Glu Ala Pro Gly 115 120 125 Phe Asn Ser Val Gly Ser Cys Pro Phe Thr Val Tyr Lys Cys Pro Pro 130 135 140 Lys Gly Leu Ser Ser Trp Gly Glu Arg Val Ala Arg Thr Ser Ala Phe 145 150 155 160 Leu Gln Ala Cys Thr Glu Lys Tyr Ser Trp Pro Glu Thr Gly Ala Glu 165 170 175 Ala Glu Leu Ser Ser Leu Arg Tyr Gln Ala Ala Arg Arg Gln Ser Ala 180 185 190 Gln Thr Thr Ala Val Ile Pro Pro Lys Asp Val Arg Glu Asp Leu Ile 195 200 205 Lys Arg Thr Thr Glu Ala Tyr Arg Ser Thr Ala Leu Pro Ala Pro Met 210 215 220 Trp Ala His Asn Phe Asp Glu Ser His Met Arg Phe Glu Phe Trp Glu 225 230 235 240 Cys Val Arg Lys Leu Lys Gly Gln Ala Gly Ser Gly Val Pro Tyr Ala 245 250 255 Ala Phe Ser Gly Arg Lys Thr Asn Asp Lys Trp Val Phe Asp His Glu 260 265 270 Ser Thr Glu Asp Leu Trp Glu Thr Val Arg Asp Arg Leu Phe Arg Leu 275 280 285 Leu Asn Gln Asp Phe Ile Asp Pro Val Gln Ala Val Lys Asp Gly Leu 290 295 300 Val Asp Pro Ile Arg Leu Phe Val Lys Leu Glu Pro His Lys Met Glu 305 310 315 320 Lys Ile Arg Asn Lys Arg Tyr Arg Leu Ile Ala Ser Val Ser Ile Val 325 330 335 Asp Gln Leu Val Ala Arg Met Leu Phe Arg Asp Gln Asn Glu Glu Glu 340 345 350 Leu Leu Gln His Met Ala Ile Pro Ser Lys Pro Gly Leu Gly Phe Ser 355 360 365 Gln Asp His Gln Val Leu Ala Phe Thr Glu Ser Val Ala Ala Leu Ala 370 375 380 Gly Thr Ser Ala Gln Asp Leu Val Asp Asn Trp Ser Arg Tyr Leu Thr 385 390 395 400 Pro Thr Asp Cys Ser Gly Phe Asp Trp Ser Val Pro Met Trp Leu Leu 405 410 415 Glu Asp Asp Leu Ala Val Arg Asn Glu Leu Thr Leu Gly Leu Pro His 420 425 430 Gly Leu Arg Lys Met Arg Glu Thr Trp Leu Lys Cys Leu Gly Gln Ser 435 440 445 Val Phe Cys Leu Ser Asn Gly Leu Leu Leu Ala Gln Thr Ser Pro Gly 450 455 460 Ile Gln Lys Ser Gly Ser Phe Asn Thr Ser Ser Thr Asn Ser Arg Met 465 470 475 480 Arg Tyr Met Leu Ala Leu Tyr Ala Gly Ala Ser Trp Ala Val Thr Met 485 490 495 Gly Asp Asp Ala Leu Glu Ser Val Gly Ser Asp Leu Ser Gln Tyr Ala 500 505 510 Arg Leu Gly Ile Lys Cys Glu Arg Ala Glu Glu Phe Asp Phe Cys Ser 515 520 525 His Leu Phe Arg Ala Pro Asp Val Val 530 535 

1. An oligonucleotide primer capable of hybridising to conserved regions of nucleic acid of carrot red leaf luteovirus (CRLV), potato leafroll polerovirus (PLRV) and at least one of barley yellow dwarf virus (BYDV) and beet western yellow virus (BWYV).
 2. A primer according to claim 1 capable of hybridising to conserved regions of nucleic acid of carrot red leaf luteovirus (CRLV), potato leafroll polerovirus (PLRV), barley yellow dwarf virus (BYDV) and beet western yellow virus (BWYV).
 3. A primer according to claim 1 or claim 2 wherein the primer hybridises to a region of nucleic acid corresponding to a RNA dependent RNA polymerase (RdRp) gene.
 4. The primer according to any one of the preceding claims wherein the primer hybridises to a region of the CRLV sequence of FIG. 1 (SEQ ID NO: 9).
 5. The primer according to any one of the preceding claims wherein the primer encodes the amino acid sequence FVKGEPH (SEQ ID NO:1) or KGEPHK (SEQ ID NO:7).
 6. The primer according to claim 5 having the nucleotide sequence 5′ TTY/GTN/AAR/GGN/GAR/CCN/CAY 3′ (SEQ ID NO:2) or AAR/GGN/GAR/CCN/CA Y/AAR/C 3′ (CL4, SEQ ID NO:8)
 7. The primer according to any one of claims 1 to 5 wherein the primer hybridise to the nucleic acid encoding amino acid sequence GFKVEV (SEQ ID NO:3) or EDDMEV (SEQ ID NO:5).
 8. The primer according to claim 8 having the nucleotide sequence 5′NAC/YTC/NAC/YTT/RAA/NCC 3′ (SEQ ID NO:4)) or 5′ CK/NAC/YTC/CAT/RTC/RTC/YTC 3′ (SEQ ID NO:6).
 9. A method for diagnosing the presence of a luteovirus sequence in a plant tissue nucleic acid sample, the method comprising the step of: hybridising a primer according to any one of claims 1 to 8 to the nucleic acid.
 10. A method according to claim 9 comprising the steps of treating the nucleic acid with a first PCR primer according to claim 1 to 9 to obtain a DNA product; treating the DNA product with one or more second PCR primers according to claim 1 to 9 to obtain a PCR product; and comparing the nucleotide sequence or corresponding amino acid sequence of the PCR product with a known nucleotide sequence or corresponding amino acid sequence characteristic of the luteovirus.
 11. A method according to claim 10 wherein said first primer is CL2 (SEQ ID NO:4) and said second primer comprises a mixture of primers CL1 (SEQ ID NO:2) and CL2 (SEQ ID NO:4).
 12. A method for diagnosing the presence of at least one of CRLV or PLRV sequences in a plant tissue, the method comprising the steps of: providing nucleic acid from the plant tissue; treating the nucleic acid with PCR primer CL2 (SEQ ID NO:4) to obtain a DNA product; treating the DNA product with PCR primers CL1 (SEQ ID NO:2) and CL3 (SEQ ID NO:6) to obtain a PCR product; and comparing a nucleotide sequence or corresponding amino acid sequence in the PCR product with a nucleotide sequence or corresponding amino acid sequence characteristic of the luteovirus to detect the presence of the luteovirus.
 13. A kit for performing the method of any one of claims 9 to 12 comprising one or more primers according to any one of claims 1 to
 8. 14. A kit according to claim 13 further comprising one or more of primers SEQ ID NOS: 12, 14 , 17 or
 18. 